Amplification and propagation of endothelial signaling is likely to be dependent upon the release of local paracrine factors. Reactive oxygen species, including superoxide derived from activated inflammatory cells, activate endothelial cells under inflammatory and ischemia/reperfusion conditions. Subsequently, endothelial damage may potentiate inflammation and ischemic organ injury. Determining a means to reduce endothelial damage is therefore warranted, and may lead to the development of novel therapeutic approaches to treat ischemic and inflammatory diseases. The long-term goal of the proposed research project is to understand the molecular mechanisms of superoxide- mediated endothelial injury. In this proposal, we postulate that superoxide is a signaling molecule that facilitates endoplasmic reticulum Ca2+ signaling and triggers mitochondrial and nuclear stress. The guiding hypothesis is that superoxide raises intracellular Ca2+ and leads to mitochondrial Ca2+ overload, resulting in mitochondrial signaling. Extracellular superoxide selectively induces Ca2+-dependent depolarization independent of other oxidant species. Further, superoxide-evoked signals activate the redox sensitive transcription factor NF-:B. The uncoupling of extracellular superoxide-evoked signal from the mitochondria results in mitochondrial membrane potential preservation and endothelial cell survival. The specific aims of this project are to examine (Specific Aim 1) how superoxide triggers upstream Ca2+ signaling and selectively activates inositol 1,4,5-trisphosphate receptors (InsP3R) both in vitro and in vivo; (Specific Aim 2) the role of extracellular superoxide-mediated Ca2+ signaling in mitochondrial pathophysiology (mitochondrial redox status-NADH and glutathione levels, mitochondrial ROS generation and mitochondrial membrane potential); (Specific Aim 3) the nuclear events that activate endothelial inflammatory mechanisms by InsP3R-linked ROS signaling. The proposed experiments should provide information on: (A) the involvement of InsP3Rs and their selective role in endothelial Ca2+ signaling during oxidative stress. (B) The relationship between mitochondrial dysfunction and inflammatory signaling in endothelial dysfunction. (C) This result will provide a novel signaling link between inflammatory and endothelial cells under pathophysiological conditions and lead to the development of novel therapeutic targets. PUBLIC HEALTH RELEVANCE The pulmonary vascular endothelium plays a key role in lung heath by translating blood signals into vascular function. During septic shock, an important and under-recognized signal is leukocyte-derived superoxide, which stimulates pulmonary endothelial cells by mobilizing calcium. Understanding the mechanisms of superoxide-mediated calcium signaling and mitochondria-nuclear stress in endothelial cells are crucial to elucidating the fundamental role of superoxide in both cell survival and death during physiological and pathological processes.